We explored the potential of mutant allele-specific gene silencing (ASGS) in providing therapeutic benefit in two established mouse models of the autosomal dominantly-inherited muscle disorders, Malignant Hyperthermia (MH) and Central Core Disease (CCD). Candidate ASGS siRNAs were designed and validated for efficacy and specificity on ryanodine receptor (RyR1) cDNA mini-constructs expressed in HEK293 cells using RT-PCR- and confocal microscopy-based assays. In vivo delivery of the most efficacious identified siRNAs into flexor digitorum brevis (FDB) muscles was achieved by injection/electroporation of footpads of 4-6 month old heterozygous Ryr1(Y524S/+) (YS/+) and Ryr1(I4895T/+) (IT/+) knock-in mice, established mouse models of MH with cores and CCD, respectively. Treatment of IT/+ mice resulted in a modest rescue of deficits in the maximum rate (∼38% rescue) and magnitude (∼78%) of ligand-induced Ca(2+) release that occurred in the absence of a change in the magnitude of electrically-evoked Ca(2+) release. Compared to the difference between the caffeine sensitivity of Ca(2+) release in FDB fibers from YS/+ and WT mice treated with SCR siRNA (EC(50): 1.1 mM versus 4.4 mM, respectively), caffeine sensitivity was normalized in FDB fibers from YS/+ mice following 2 (EC(50): 2.8 mM) and 4 week (EC(50): 6.6 mM) treatment with YS allele-specific siRNA. Moreover, the temperature-dependent increase in resting Ca(2+) observed in FDB fibers from YS/+ mice was normalized to WT levels after 2 weeks of treatment with YS allele-specific siRNA. As determined by quantitative real time PCR, the degree of functional rescue in YS/+ and IT/+ mice correlated well with the relative increase in fractional WT allele expression.

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Central Core Disease (CCD) and Malignant Hyperthermia (MH) are linked to single amino acid substitutions in the skeletal muscle Ca2+ release channel, the type 1 ryanodine receptor (RyR1). We focus on two autosomal dominant (AD) RyR1 mutations, Y522S (YS) and I4898T (IT), which cause MH and CCD, respectively. The AD mode of inheritance and data indicating knock-out of one RyR1 allele is well-tolerated in mice led us to hypothesize that allele-specific gene silencing (ASGS) of the mutant allele would rescue RyR1 functional defects in skeletal muscle cells from YS and IT knock-in mice. We evaluated the functional consequences of allele-specific silencing in YS and IT muscle cells using short interfering RNAs (siRNAs). To screen potential siRNAs for relative knockdown efficacy and allele specificity, we generated cDNAs encoding fusion proteins derived from wild type (WT) (Venus-Exons-3XFLAG) and either YS or IT mutation-containing (Cherry-Exons-3XHA) exons. Simultaneous transfection of these cDNAs and siRNAs into HEK293 cells and subsequent evaluation of mRNA (semi-quantitative RT-PCR) and protein levels (fluorescence microscopy and western blotting) was used to determine knockdown efficacy and allele-specificity prior to functional rescue experiments. Myotubes derived from heterozygous YS mice (YS/+) exhibit ∼4-fold increase in caffeine sensitivity (EC50 values were 0.5mM and 2.3mM for YS/+ and WT, respectively). Treatment with a YS-selective siRNA, normalized caffeine sensitivity (EC50 = 2.5mM) without decreasing peak caffeine-induced release. Similarly, YS-selective siRNA treatment rescued the increased voltage sensitivity of Ca2+ release in YS/+ myotubes determined in perforated-patch clamp experiments (VF1/2: WT = −18mV, YS/+ scrambled = −35mV, YS/+ YS-selective = −18mV). These results indicate that ASGS represents a promising approach for normalization of RyR1 function in MH and CCD. Similar functional rescue experiments in adult skeletal muscle fibers are currently underway.

The RYR1 functions as the Ca release channel in the skeletal muscle SR. The functional RYR1 SR Ca release channel is a 2.3-megadalton homomeric assembly of four 565-kD RYR1 subunits. Each RYR1 subunit is composed of a large N-terminal cytosolic “foot” region and six to eight transmembrane sequences located within the C-terminal portion of the protein (Du et al., 2002, 2004). By analogy with known K channel structures, the selectivity filter of the RYR1 Ca release channel is determined by a conserved hydrophobic sequence Gly-Ile-Gly (amino acids 4894-4895-4896 in mouse RYR1) (Zhao et al., 1999; Gao et al., 2000; Williams et al., 2001) located between the final two transmembrane domains. Fully assembled tetrameric Ca release channels

Summary The I4898T (IT) mutation in type 1 ryanodine receptor (RyR1), the Ca 2+ release channel of the sarcoplasmic reticulum (SR) is linked to a form of central core disease (CCD) in humans and results in a nonleaky channel and excitation–contraction uncoupling. We characterized age‐dependent and fiber‐type‐dependent alterations in muscle ultrastructure, as well as the magnitude and spatiotemporal properties of evoked Ca 2+ release in heterozygous Ryr1 I4895T/WT (IT/+) knock‐in mice on a mixed genetic background. The results indicate a classical but mild CCD phenotype that includes muscle weakness and the presence of mitochondrial‐deficient areas in type I fibers. Electrically evoked Ca 2+ release is significantly reduced in single flexor digitorum brevis (FDB) fibers from young and old IT/+ mice. Structural changes are strongly fiber‐type specific, affecting type I and IIB/IIX fibers in very distinct ways, and sparing type IIA fibers. Ultrastructural alterations in our IT /+ mice are also present in wild type, but at a lower frequency and older ages, suggesting that the disease mutation on the mixed background promotes an acceleration of normal age‐dependent changes. The observed functional and structural alterations and their similarity to age‐associated changes are entirely consistent with the known properties of the mutated channel, which result in reduced calcium release as is also observed in normal aging muscle. In strong contrast to these observations, a subset of patients with the analogous human heterozygous mutation and IT/+ mice on an inbred 129S2/SvPasCrl background exhibit a more severe disease phenotype, which is not directly consistent with the mutated channel properties.

The skeletal muscle ryanodine receptor plays a crucial role in excitation–contraction (EC) coupling and is implicated in various congenital myopathies. The periodic paralyses are a heterogeneous, dominantly inherited group of conditions mainly associated with mutations in the SCN4A and the CACNA1S genes. The interaction between RyR1 and DHPR proteins underlies depolarization-induced Ca2+ release during EC coupling in skeletal muscle. We report a 35-year-old woman presenting with signs and symptoms of a congenital myopathy at birth and repeated episodes of generalized, atypical normokalaemic paralysis in her late teens. Genetic studies of this patient revealed three heterozygous RYR1 substitutions (p.Arg2241X, p.Asp708Asn and p.Arg2939Lys) associated with marked reduction of the RyR1 protein and abnormal DHPR distribution. We conclude that RYR1 mutations may give rise to both myopathies and atypical periodic paralysis, and RYR1 mutations may underlie other unresolved cases of periodic paralysis with unusual features.

The mutation from isoleucine to threonine of the skeletal isoform of the ryanodine receptor (RyR1) at residue 4898 results in severe Central Core Disease (CCD). Under homozygous expression (IT/IT), we reported a lack of Ca2+ release in response to electrical and pharmacological activation despite SR Ca2+ store content indistinguishable from control. Here we used heterozygous knock-in mice for the I4895T (IT/+; analogous to human I4898T) RyR1 mutation to determine the effects of the mutation on muscle strength and Ca2+ handling in flexor digitorum brevis (FDB) and interosseous muscle fibers. We compared in vivo muscle strength of wild-type (WT) and IT/+ mice. IT/+ mice exhibited significant weakness in both upper body and grip strength assays (4-paw peak grip force: 2400 ± 70 mN, n=8 and 2040 ± 80 mN, n=14 in WT and IT/+ mice, respectively). We also determined the magnitude of action potential- and ligand-evoked Ca2+ release in single intact FDB fibers using Ca2+ fluorometry. The magnitude of both electrically- and ligand-evoked Ca2+ release was significantly reduced in IT/+ fibers. Moreover, the maximum rate of change in mag-fluo-4 fluorescence during the rising phase of the electrically-evoked Ca2+ transient was significantly reduced in IT/+ fibers (WT 0.17 ± 0.01 ΔF/F/ms vs IT/+ 0.11 ± 0.01 ΔF/F/ms, n= 53, 56, respectively). Finally, the frequency (1.9 ± 0.5 and 0.8 ± 0.3 events/scan) and Ca2+ spark mass (5.9 ± 0.3 and 4.6 ± 0.2 μm3) of local Ca2+ release induced by osmotic shock (440 mOsm with sucrose, 750 lines/sec) were reduced in acutely dissociated IT/+ interosseous fibers compared to that of WT fibers. Together, these findings are consistent with the hypothesis that the IT mutation in the putative RyR1 selectivity filter significantly reduces Ca2+ flux through the channel.

Voltage-dependent calcium channels (Ca V ) open in response to changes in membrane potential, but their activity is modulated by Ca 2+ binding to calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal differences between structures cannot adequately describe CaM's role in the regulation of these channels. We report a unique crystal structure of a 77-residue fragment of the Ca V 1.2 α 1 subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ domains, in complex with Ca 2+ ·CaM in 2 distinct binding modes. The structure of the Ca V 1.2 fragment is an unusual dimer of 2 coiled-coiled pre-IQ regions bridged by 2 Ca 2+ ·CaMs interacting with the pre-IQ regions and a canonical Ca V 1-IQ–Ca 2+ ·CaM complex. Native Ca V 1.2 channels are shown to be a mixture of monomers/dimers and a point mutation in the pre-IQ region predicted to abolish the coiled-coil structure significantly reduces Ca 2+ -dependent inactivation of heterologously expressed Ca V 1.2 channels.

A heterozygous Ile4898 to Thr (I4898T) mutation in the human type 1 ryanodine receptor/Ca 2+ release channel (RyR1) leads to a severe form of central core disease. We created a mouse line in which the corresponding Ryr1 I4895T mutation was introduced by using a “knockin” protocol. The heterozygote does not exhibit an overt disease phenotype, but homozygous (IT/IT) mice are paralyzed and die perinatally, apparently because of asphyxia. Histological analysis shows that IT/IT mice have greatly reduced and amorphous skeletal muscle. Myotubes are small, nuclei remain central, myofibrils are disarranged, and no cross striation is obvious. Many areas indicate probable degeneration, with shortened myotubes containing central stacks of pyknotic nuclei. Other manifestations of a delay in completion of late stages of embryogenesis include growth retardation and marked delay in ossification, dermatogenesis, and cardiovascular development. Electron microscopy of IT/IT muscle demonstrates appropriate targeting and positioning of RyR1 at triad junctions and a normal organization of dihydropyridine receptor (DHPR) complexes into RyR1-associated tetrads. Functional studies carried out in cultured IT/IT myotubes show that ligand-induced and DHPR-activated RyR1 Ca 2+ release is absent, although retrograde enhancement of DHPR Ca 2+ conductance is retained. IT/IT mice, in which RyR1-mediated Ca 2+ release is abolished without altering the formation of the junctional DHPR-RyR1 macromolecular complex, provide a valuable model for elucidation of the role of RyR1-mediated Ca 2+ signaling in mammalian embryogenesis.